This is a competitive renewal of an R24 grant focused on SIRT3, the major mitochondrial protein deacetylase. Our initial funding was limited to two years due to NIH budget constraints, but we believe that during the first 18 months of this grant we have made great progress in forming and an interdisciplinary team that has defined novel biological functions of SIRT3 and its role in regulation of systemic metabolism. We have found that SIRT3 expression is differentially regulated in liver and muscle in fasting and diabetes. In liver, increased SIRT3 during fasting leads to deacetylation of key mitochondrial enzymes in the fatty acid oxidation pathway and to an increase in their enzymatic activities. In contrast, SIRT3 expression decreases in muscle during fasting, leading to hyperacetylation of pyruvate dehydrogenase and several components of the electron transport chain. Metabolomic analysis of SIRT3KO reveals possible tissue-specific differences in regulation of fatty acid, glucose, and amino acid oxidation that will be explored further in the current application. These studies clearl show that SIRT3KO mice have defective fatty acid oxidation in liver and reduced metabolic flexibility and reduced ATP levels in muscle, as well as insulin resistance due to increased ROS and activation of stress kinases. Furthermore, SIRT3KO mice placed on a high fat diet show accelerated development of a syndrome that mimics human metabolic syndrome with obesity, type 2 diabetes, lipid abnormalities, and steatohepatitis. Together these findings point to a role of reversible mitochondrial protein acetylation as a key regulator of mitochondrial metabolism and SIRT3 as a potentially important factor in the pathogenesis of type 2 diabetes and the metabolic syndrome. The overall goal of this proposal is to extend these studies to completely define the role of protein acetylation and SIRT3 in mitochondrial function and control of metabolism. We will take advantage of our highly collaborative and multidisciplinary team harnessing the power of mass spectrometry-based proteomics, metabolomics, molecular biology, extensive physiological testing and unique animal models to further expand our understanding of this important process in regulation of mitochondrial function and metabolism.